HIV requires host machinery to replicate. In turn, the host cell protects itself using antiviral factors. A full understanding of the host factors involve in HIV replication will provide us with valuable knowledge of HIV virology and cell biology. It will also help to develop new anti-HIV therapy. Functional screens using genome-wide siRNA libraries have implicated hundreds of host proteins as modulators of HIV replication. In this proposed project, we will use multiple RNAi resources to identify the innate host proteins that restrict HIV-1 replication. The remaining goals for this project are to focus on further validation and functional study of the restriction factors that come out of screens. Aim 1 is the validation and functional analysis of HIV-1 restriction factors identified through genome-wide siRNA screens. We recently completed siRNA screens using multiple RNAi resources (three orthologous siRNA libraries). The screens identified 212 potential host restriction factors. For this aim, we plan to focus on a shortened list of potential anti-HIV host factors, and perform further validation and functional studies to confirm their roles. The plan includes confirmation of siRNA knockdown efficacy, RNAi effect rescue, validation in human primary cells, and mechanistic studies of a few of promising HIV-1 restriction factors. In addition, we plan to test the inhibitory efforts of confirmed restriction factors on other viruses. Aim 2 is the identificatin of factors that mediate interferon- induced restriction of HIV-1 through genome-wide siRNA screens. It has been long known that Type I interferon (IFN) can induce the expression of a large set of interferon-stimulated genes and establish a potent antiviral response. Both early and late steps of HIV-1 replication can be blocked by IFN-1. In this aim, we will perform genome-wide siRNA screens to identify HIV-1 restriction factors whose activity is required by IFN. The candidate restriction factors will be further filtered by performing comprehensive bioinformatic analysis. A set of potential IFN-induced HIV-1 restriction factors will be selected for further validation and functional studies. We believe that our study of HIV-1 restriction factors will provide new insights into the barriers to viral infection and may have important ramifications for the identification of new antiviral targets. PUBLIC HEALTH RELEVANCE: Although there are several effective drugs for treating AIDS/HIV that can slow the course of the disease, there is still no cure or vaccine. We propose to use functional genomic approaches to identify novel HIV restriction factors, and further validate and study their function in physiologically relevant, HIV-infected host cells. The discovery and functional studies of novel HIV restriction factors will help unravel the previously unrecognized cellular antiviral mechanisms that can be used in new strategies to prevent or treat HIV infection.